1. Discovery of Alternative Splicing
First discovered with an Immunoglobulin heavy
chain gene (D. Baltimore et al.)
Alternative splicing gives two forms of the protein with different C-termini
1 form is shorter and secreted
Other stays anchored in the plasma membrane via C-terminus
~40% of human genes produce alternatively spliced transcripts!

2. Alternative splicing of the mouse immunoglobulin μ heavy chain gene
The M exons are not spliced to the secreted terminus exon (C) in the mRNA that codes for the membrane-bound form of the protein.
S-signal peptide C - constant region
V- variable region green – membrane anchor
Red- untranslated reg. yellow – end of coding reg. for secreted form
Fig

3. Regulation of Alternative splicing
Sex determination in Drosophila involves 3 regulatory genes that are differentially spliced in females versus males; 2 of them affect alternative splicing
Sxl (sex-lethal) - promotes alternative splicing of tra (exon 2 is skipped) and of its own (exon 3 is skipped) pre-mRNA
Tra – promotes alternative splicing of dsx (last 2 exons are excluded)
Dsx (double-sex) - Alternatively spliced form of dsx needed to maintain female state
Fig

4. Alternative splicing in Drosophila maintains the female state.
The female Sxl protein promotes alternative splicing of its own mRNA and the tra mRNA.
Tra plus Tra-2 promote alternative splicing of dsx.
Female version of Dsx maintains the female state by repressing male-specific genes, and vice-versa to get male flies.
Sxl and Tra are SR proteins!
Tra and Tra-2 bind a repeated element in exon 4 of dsx mRNA, causing it to be retained in mature mRNA.
Fig

5. Trans-Splicing (Ch. 16.3) Intermolecular splicing of pre-mRNAs
First discovered in African trypanosomes, a disease(African Sleeping Sickness)-causing parasitic protozoan.
The mRNAs had 35 nt not encoded in the main gene – called the spliced leader sequence.
Spliced leader (SL) is encoded separately, and there about 200 copies in the genome .
SL primary transcript contains ~100 nt that resemble the 5’ end of a NmRNA intron.

6. Organisms that trans-splice nuclear genes.
from Fig. 16.8
Schistosoma & Ascaris are parasitic worms. Trans-splicing in organelles involves mostly group II but also a group I intron.
Trypanosome Schistosoma Ascaris Euglena
Trans-splicing also occurs in plant chloroplast and mitochondrial genes!

7. 2 possible models to explain the joining of the SL to the coding region of a mRNA
1. Primed transcription by SL
2. Trans-splicing model
Fig. 16.9

8. Trans-splicing in TrypanosomesSL
Trans-splicing should yield some unique “Y –shaped” intron-exon intermediates containing the SL half-intron.
Fig

9. Release of the SL half-intron from larger RNAs by a debranching enzyme.
32P-labeled total or polyA RNA was treated with debranching enzyme and then electrophoresed. The 147-nt RNA is probably 5S rRNA. The RNA of ~ nt is the SL RNA.
This result is consistent with a trans-splicing model rather than a cis-splicing mechanism.
Figs , 16.12

10. Some of these organisms (Trypanosomes and Euglena) also have polycistronic genes.
Section 16.3 and Figure in Weaver, 3rd edition, has info on Trypanosomes.
Trypanosome Schistosoma Ascaris Euglena
Parasitic Worms
Fig. 16.8

11. Cap stimulates splicing of the first intron in a multi-intron pre-mRNA
32P-labeled substrate RNAs were incubated in a Hela nuclear extract.
RNA = Exons 13, 14 and 15 of delta-crystallin pre-mRNA (plus the introns). Band a lariat form of intron 1.
Splicing of 1st intron very poor with uncapped pre-mRNA.
May have been methylation of Cap in extract.
Fig

12. CAP Binding Complex (CBP)
Contains 2 proteins of 80 (CBP80) and 20 (CBP20) kiloDaltons
Depletion of CBP from a splicing extract using antibody against CBP80 inhibited splicing of the first intron in a model pre-mRNA
Further analysis showed an inhibition of spliceosome formation
CBP may be important for spliceosome formation in vivo on first intron

13. Poly A-Dependent Splicing of the Last Intron in a 2-intron pre-RNA
Double-spliced mRNA
Splicing of the 2nd intron in this pre-mRNA is reduced by a mutation in the polyadenylation signal (wild-type hexamer=AAUAAA).
Splicing of the 1st intron is normal.
Fig

14. RNA Splicing and Disease
~ 15 % of the mutations that cause genetic diseases affect pre-mRNA splicing.
Many are cis-acting mutations at the splice-sites, the branch point, or sequences that promote (enhancers) or inhibit (silencers) splicing of certain exons.
OMIM (Online Mendelian Inheritance in Man) - database of human genetic mutations and disorders at NCBI, a.k.a. the National Center for Biotechnology Information) (link on Blackboard)